This application is a competitive renewal for 1R01HL103866 entitled Gut flora metabolism of dietary phosphatidylcholine and cardiovascular disease. Over the past funding period we discovered that foods associated with increased cardiovascular disease (CVD) risks like meats, egg yolk and high-fat dairy products may contribute to enhanced development of CVD by an unrecognized gut microbial (gut flora) pathway that impacts host cholesterol and sterol metabolism, engendering enhanced susceptibility for atherosclerosis development, and cardiovascular disease risks. The pathway begins with dietary trimethylamine containing nutrients abundant in these foods (phosphatidylcholine, choline, and L-carnitine), and an obligatory gut microbial pathway that ultimately leads to formation of an atherogenic compound, trimethylaine-N-oxide (TMAO). Using basic, translational and human clinical studies, we show that gut flora participate in development of CVD and its adverse events. In unpublished studies we also see a mechanistic link between gut flora and enhanced platelet function and development of a pro-thrombotic phenotype, a critical feature of increased risk for thrombotic events like heart attack, stroke and death. We propose to further explore the nature of gut flora involvement in development of atherosclerosis and a pro-thrombotic phenotype in vivo. We will do so through two Specific Aims: (i) Testing gut microbiota involvement in atherosclerosis and a pro-thrombotic phenotype; and (ii) Testing if gut microbiota choline trimethylamine lyase pathway involved in TMAO formation is a viable therapeutic target for reducing atherosclerosis and a pro-thrombotic phenotype. Successful completion of the proposed studies will change the way in which complex traits like atherosclerosis and thrombosis are considered. They will unambiguously define a role for gut flora in CVD development and its major adverse complications MI, stroke and death. They also will evaluate the clinical utility of both TMAO as a diagnostic test in the setting of acute coronay syndromes, and the dietary or pharmacologic targeting of microbial TMA/TMAO production for the treatment and prevention of CVD.